Heparin/heparan sulfate (HS) is a linear polymer covalently attached to the protein cores of proteoglycans, which are abundant and ubiquitously expressed in almost all animal cells. HS is assembled by the action of a large family of enzymes that catalyze the following series of reactions: chain polymerization comprising the alternating addition of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) residues; GlcNAc N-deacetylation and N-sulfation; glucuronic acid epimerization to L-iduronic acid (IdoUA); 2-O-sulfation of uronic acid residues; and 3-O- and 6-O-sulfation of glucosaminyl residues.
The interaction between HS and various proteins occur in highly sulfated regions of the HS. Furthermore, the specificity of any HS-protein interaction is largely dictated by arrangement of sulfates along the HS chain. For example, the pentasaccharide sequence, GlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S, represents the minimum sequence required for antithrombin (AT) binding, where the 3S (3-O-sulfate) and 6S (6-O-sulfate) groups constitute the most critical elements involved in the binding (12-16). The AT-HS complex has potent anticoagulant properties. Several enzymes involved in anticoagulant heparan sulfate (HSact) biosynthesis have been purified and cloned. For example, glucosaminyl 3-O sulfotransferase (3-OST) and glucosaminyl 6-O-sulfotransferase (6-OST) proteins have been purified and cloned (17,18). Multiple isoforms of 6-OST and 3-OST proteins have been isolated and shown to have tissue-specific expression patterns and distinct substrate specificities.
Two different sulfated domains are present in HS, namely, the NS domain and NAc/NS domain (40,41). The NS domains consist of contiguous iduronosyl N-sulfoglucosamine units, while the NAc/NS domain consists of alternating N-acetylated and N-sulfated disaccharides. Acceptor specifcities of 6-OST-1, 6-OST-2, and 6-OST-3 using N-sulfated heparosan and desulfated re-N-sulfated heparin as substrate, indicated that the sulfation of position 6 of the N-sulfoglucosamine residues in the NS domain is catalyzed by 6-OST-1, 2A, 2B, and 3 and the sulfation of position 6 of the N-sulfoglucosamine residues in the NA/NS domain are catalyzed by 6-OST-2 and 6-OST-3 (2).
Tissue-specific and developmentally regulated expression of the HS biosynthetic enzymes and enzyme isoforms produce HS chains with specific sequences (1-3). This microheterogeneity enables HS to interact with a broad array of protein ligands that modulate a wide range of biological functions in development, differentiation, homeostasis, and bacterial/viral entry (reviewed in refs (4-11)). Synthetic polysaccharides which possess such specific sequences may be used to modulate such biological functions.
Heparin preparations, particularly preparations comprising HSact, have been used clinically as anticoagulant therapeutics for the prevention and treatment of thrombotic disease. HSact preparations have also been used to maintain blood fluidity in extracoporeal or corporeal medical devices such as dialysis devices and stents, respectively.